This invention relates to the field of enzymatic assays and, in particular, assays for protein kinase activity involving modified fluorescent proteins.
Protein phosphorylation is one of the most important general mechanisms of cellular regulation. Protein phosphorylation commonly occurs on three major amino acids, tyrosine, serine or threonine, and changes in the phosphorylation state of these amino acids within proteins can regulate many aspects of cellular metabolism, regulation, growth and differentiation. Changes in the phosphorylation state of proteins, mediated through phosphorylation by kinases, or dephosphorylation by phosphatases, is a common mechanism through which cell surface signaling pathways transmit and integrate information into the nucleus. Given their key role in cellular regulation, it is not surprising that defects in protein kinases and phosphatases have been implicated in many disease states and conditions. For example, the over-expression of cellular tyrosine kinases such as the EGF or PDGF receptors, or the mutation of tyrosine kinases to produce constitutively active forms (oncogenes) occurs in many cancer cells. Drucker et al. (1996) Nature Medicine 2: 561-56. Protein tyrosine kinases are also implicated in inflammatory signals. Defective Thr/Ser kinase genes have been demonstrated to be implicated in several diseases such as myotonic dystrophy as well as cancer, and Alzheimer's disease (Sanpei et al. (1995) Biochem. Biophys. Res. Commun. 212: 341-6; Sperber et al (1995) Neurosci. Lett. 197: 149-153; Grammas et al (1995) Neurobiology of Aging 16: 563-569; Govoni et al. (1996) Ann. N.Y. Acad. Sci. 777: 332-337).
The involvement of protein kinases and phosphatases in disease states makes them attractive targets for the therapeutic intervention of drugs, and in fact many clinically useful drugs act on protein kinases or phosphatases. Examples include cyclosporin A which is a potent immunosuppressant that binds to cyclophilin. This complex binds to the Ca/calmodulin-dependent protein phosphatase type 2B (calcineurin) inhibiting its activity, and hence the activation of T-cells. (Sigal and Dumont (1992), Schreiber and Crabtree (1992)). Inhibitors of protein kinase C are in clinical trails as therapeutic agents for the treatment of cancer. (Clin. Cancer Res. (1995) 1:113-122) as are inhibitors of cyclin dependent kinase. (J. Mol. Med. (1995) 73:(10):509-14.)
The number of known kinases and phosphatases are growing rapidly as the influence of genomic programs to identify the molecular basis for diseases have increased in size and scope. These studies are likely to implicate many more kinase and phosphatase genes in the development and propagation of diseases in the future, thereby making them attractive targets for drug discovery. However current methods of measuring protein phosphorylation have many disadvantages which prevents or limits the ability to rapidly screen using miniaturized automated formats of many thousands of compounds. This is because current methods rely on the incorporation and measurement of .sup.32 P into the protein substrates of interest. In whole cells this necessitates the use of high levels of radioactivity to efficiently label the cellular ATP pool and to ensure that the target protein is efficiently labeled with radioactivity. After incubation with test drugs, the cells must be lysed and the protein of interest purified to determine its relative degree of phosphorylation. This method requires high numbers of cells, long preincubation times, careful manipulation and washing steps (to avoid artifactual phosphorylation or dephosphorylation), as well as a method of purification of the target protein. Furthermore, final radioactive incorporation into target proteins is usually very low, giving the assay poor sensitivity. Alternative assay methods, for example based on phosphorylation-specific antibodies using ELISA-type approaches, involve the difficulty of producing antibodies that distinguish between phosphorylated and non-phosphorylated proteins, and the requirement for cell lysis, multiple incubation and washing stages which are time consuming, complex to automate and potentially susceptible to artifacts.
Kinase assays based on purified enzymes require large amounts of purified kinases, high levels of radioactivity, and methods of purification of the substrate protein away from incorporated .sup.32 P-labelled ATP. They also suffer from the disadvantage of lacking the physiological context of the cell, preventing a direct assessment of a drugs toxicity and ability to cross the cells plasma membrane.
Fluorescent molecules are attractive as reporter molecules in many assay systems because of their high sensitivity and ease of quantification. Recently, fluorescent proteins have been the focus of much attention because they can be produced in vivo by biological systems, and can be used to trace intracellular events without the need to be introduced into the cell through microinjection or permeabilization. The green fluorescent protein of Aequorea Victoria is particularly interesting as a fluorescent indicator protein. A cDNA for the protein has been cloned. (D. C. Prasher et al., "Primary structure of the Aequorea Victoria green-fluorescent protein," Gene (1992) 111:229-33.) Not only can the primary amino acid sequence of the protein be expressed from the cDNA, but the expressed protein can fluoresce. This indicates that the protein can undergo the cyclization and oxidation believed to be necessary for fluorescence. The fluorescence of green fluorescent protein is generated from residues S65-Y66-G67.
Fluorescent proteins have been used as markers of gene expression, tracers of cell lineage and as fusion tags to monitor protein localization within living cells. (M. Chalfie et al., "Green fluorescent protein as a marker for gene expression," Science 263:802-805; A. B. Cubitt et al., "Understanding, improving and using green fluorescent proteins," TIBS 20, November 1995, pp. 448-455. U.S. Pat. No. 5,491,084, M. Chalfie and D. Prasher. Furthermore, mutant versions of green fluorescent protein have been identified that exhibit altered fluorescence characteristics, including altered excitation and emission maxima, as well as excitation and emission spectra of different shapes. (R. Heim et al., "Wavelength mutations and posttranslational autoxidation of green fluorescent protein," Proc. Natl. Acad. Sci. USA, (1994) 91:12501-04; R. Heim et al., "Improved green fluorescence," Nature (1995) 373:663-665.) These properties add variety and utility to the arsenal of biologically based fluorescent indicators.
There is a need for assays of protein phosphorylation that are simple, sensitive, non-invasive, applicable to living cells and tissues and that avoid the use of any radioactivity.